Target-based drug discovery approaches have traditionally used steady-state affinity as the main parameter to assess a compound activity and predict its performance in relevant biological models. The majority of drug discovery projects rely upon estimates of compound affinity to a target protein to guide medicinal chemistry in early stages. However, evidence is plentiful that compounds with the same affinity but very different on-(Kon) and off-rates (Koff) can have a very different biological activity profile. Many experts recognize kinetic binding data as a decisive element in drug discovery that directly impact drug efficacy and safety (Copeland 2006; Swinney 2009; Mosnma 2010). For instance, compounds displaying diffusion-controlled association rates are expected to be more efficient thrombin inhibitors (Elg 1997) and transient kinetic can also be an advantage in the development of antipsychotic drugs, where mechanism-based toxicity can occur if the target receptor is inhibited for a long period of time (Tresadern 2011). On the other hand, the selectivity and efficacy of drugs with longer residence time than their plasma half-lives are likely to be underestimated by classical pharmacokinetic/pharmacodynamic models, and the inclusion of binding kinetic data is expected to really improve the predictive value of these models (Dahl 2013).
There are a variety of instruments and measurement techniques for kinetic analysis. Classical methods such as stopped flow, jump dilution and radioligand binding competition assays are tedious and cumbersome, mostly of limited throughput. There are many examples of kinetic characterization by using radiolabel ligands, most of them using radioligand competition assays and the mathematical model described by Motulski and Mahan in the eighties (Motulski 1984). To perform these assays, commonly a titrated ligand analog to the natural substrate is synthesized and custom-labeled. This radioactive ligand is first characterized kinetically to determine its association and dissociation rates. Then, unknown ligand (non-radioactive) plus the radioactive ligand are added simultaneously to the reaction mixture, and the reaction is leaved for different times. After stopping reactions, separation of bound from free radioligand is performed by rapid filtration techniques (as GF/B filter plate using a FilterMate harvester, PerkinElmer) and filters must be extensively washed before adding the scintillation cocktail. Filter-bound radioactivity is further measured by scintillation spectrometry, using conventional scintillation counters. As can be shown, these methods are tedious and time-consuming, and several incubating and washing steps are needed before the final read-out of each time point. Moreover, special care it is needed to work, store and destroy radiolabeled compounds.
Recently, some TR-FRET binding methods have been adapted to measure off-rates (Koff), reporting binding events in real time and using the classical large dilution method (LanthaScreen™_binding and Transcreener™). Nevertheless, these methods rely on previous knowledge of the affinity of the drug target interaction, which must be previously determined in classical titration experiments with different concentration of inhibitors. Then, every molecule must be pre-incubated with the target at a concentration between 10 to 40 times its Ki, assuring that all binding sites are occupied by the inhibitor (>90%). As the affinity of a molecule against its target is an intrinsic feature of each molecule, the affinities of different molecules vary in a broad range. To automatize this process, it is necessary to use sophisticated liquid handling systems able to pick-up different volumes from different plate positions from a first plate and subsequently dispensing those volumes in specific wells of a second plate (the so-called called “cherry-picking). Moreover, analyzing compounds with very different affinities in parallel, necessarily involve the need to make intermediate dilutions of the most potent compounds. In some cases, it may be also mandatory to use intermediate dilution plates to avoid excessive cost associated with large volumes. As it can be easily deduced, the above protocol is not useful to reach kinetic analysis in a high-throughput format. This is one of the main reasons why the kinetic profile of molecules is traditionally done only with selected molecules that have already successfully advance in the drug-discovery process (molecules that are working in an appropriate way in animal models or in clinical trials).
Other kinetic methods rely on label-free biophysical techniques, including isothermal titration calorimetry (ITC), nuclear magnetic resonance, mass spectrometry and biosensors, among others. ITC calls for large quantities of purified proteins and allows to moderate throughput despite the technical advancements. Therefore, Biosensor-based techniques, with Surface Plasmon Resonance (SPR) being the most prominent one, are the preferred techniques for kinetic characterization of drug candidates. The SPR based systems enable the detection and quantification of biological interactions in real time, without the use of labels (Lieberg 1983). Target of interest must be immobilized on the surface's chip and then, the analyte is injected through the system. The target immobilization is a critical step in the development of reliable SPR assays and although a variety of sensor surfaces are available and a broad range of techniques can be used for ligand immobilization, SPR is still restricted to membrane proteins, since these proteins are not robust enough to endure immobilization on a chip surface. Moreover, the immobilized targets are not in their native way, and some binding sites may be not accessible, eluding the binding of the ligand. In addition, SPR involves expensive laboratory equipment and demands highly trained users, which may difficult to be a general method to assess kinetic profiling in a high throughput format.
As can be outlined, there is an increasing demand for improved methods and technologies that enable accurate, cost-effective and high throughput measurements of drug-target association and dissociation rates. There is a need for a reliable, robust and sensitive platform aimed to analyze the massive kinetic profile of new molecules against its main and other potential targets.
Here we describe a robust and sensitive platform aimed to determine the massive kinetic profile of new molecules against its main target (Binding Kinetics Profiling) and also against other related targets (Kinetic Selectivity Profiling) in a high-throughput format. The platform of the invention combines the versatility of radio-ligand binding assays with the advantages of new homogeneous assays based on fluorescent probes, thus saving time and costs, and also protecting the environment.
The access to kinetic data at the early stages of the discovery process will create great opportunities for a much improved early drug discovery paradigm. In fact, such considerations have guided the first reported examples of lead compounds and clinical drug candidates selected during early stages on the basis of their target binding kinetics (Langlois 2012).